Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity

Definitions

• the present invention relates to an RPDL protein which is a novel transcriptional control protein, a process for producing this protein, a method of using the same, a DNA encoding the protein, and a gene analysis method using the DNA.
• the present invention finds applications in the pharmaceutical field.
• genes execute selective expression, for example, at a specific time or site or when a certain stimulus has been given.
• the expression of the genes involves two important steps consisting of producing a mRNA on the basis of information stored in the DNA sequence (transcription) and producing a protein by the action of the mRNA (translation).
• transcriptional control proteins proteins
• Analyzing in detail the mechanism of the above transcriptional control is a task extremely important from the viewpoint of learning the selective expression control mechanism of genes, namely, the cell differentiation or amplification or various gene activities and ultimately the fundamental system relating to, for example, life and death. It is expected that the analysis of the mechanism of the above transcriptional control would break through difficult problems of not only tumors but also other various diseases and abnormalities, and further aging, dementia, obesity, etc.
• Known transcriptional control proteins include those specific for some genes and those commonly acting on a wide variety of genes. From the viewpoint of function, the known transcriptional control proteins include not only those capable of activating the transcription or inactivating the same but also those having both of the above capabilities [see M. Ptashne, Scientific American, Vol.260, p.p.40-47 (1989)].
• isolating a human gene encoding the above important transcriptional control protein and identifying the protein has an extremely important significance in that a marked progress can be realized in the direct elucidation of the transcriptional control mechanism of the cells of multicellular organisms having such aspects as development, differentiation and tissue, especially, human per se.
• the yeast transcription factor RPD3 controls not only the transcription of high- and low-affinity potassium transporter gene TRK2 but also the transcription of many genes including genes PHO5, STE6 and TY2 as the target. Further, it is known that the yeast PRD3 protein has both the functions of activation and inactivation [see M. Vidal and R.F. Gaber, Mol. Cel. Biol. Vol.11, p.p.6317-6327 (1991)].
• the present inventors have determined the 5'-terminal nucleotide sequence of each clone derived from a cDNA library prepared from human fetal lung and have found a clone exhibiting homology with the sequence of the RPD3 gene of a yeast. Further, they have determined the DNA sequence of this clone and have obtained a full-length cDNA encoding a novel protein. It has been confirmed that the amino acid sequence of the protein encoded by this cDNA exhibits a significant similarity to that of the yeast RPD3 and this protein is a novel human transcriptional control protein that has never been reported.
• the present inventors have confirmed that the gene encoding this protein is an important gene which has expressed in all the studied human tissues excluding the brain by a gene analysis according to the Northern blotting technique using the above cDNA as a probe.
• the present inventors have confirmed that the gene encoding this protein is localized at 1p34.1 on the short arm of the chromosome 1, the region where a deletion is recognized in mammary and gastric carcinomas, by the chromosomal mapping according to the FISH (fluorescent in situ hybridization) technique using the above cDNA as a probe.
• FISH fluorescent in situ hybridization
• the present invention has enabled not only the production of a transformant having, introduced thereinto, the cDNA encoding the above human transcriptional control protein or a DNA obtained by artificially mutating the same by introducing the cDNA or the DNA into a host such as E. coli, yeast, an insect cell and a mammal cell, but also the production of the above protein or its variant with the use of the transformant and further the production of an antibody capable of binding with the above protein or its variant.
• the present invention has enabled, on the level of human cells, not only the analyses of the interaction between the above protein and other factors capable of binding therewith, human genes controlled as the target and the activation and inactivation functions of the above protein as the transcriptional control factor, but also studies of the effects caused by the mutation of the DNA encoding the above protein.
• the present invention provides an RPDL protein having an amino acid sequence, said amino acid sequence comprising the whole or a part of the amino acid sequence specified in sequence ID NO 1, or a variant of said RPDL protein.
• the variant of said RPDL protein is one of RPDL proteins, has an amino acid sequence comprising the whole or a part of an amino acid sequence which is identical with the one specified in sequence ID NO 1 except that one or more amino acids are added thereto, deleted therefrom or inserted thereinto, or that one or more amino acids are substituted for one or more amino acids contained in sequence ID NO 1, and acts in the same manner as that of said RPDL protein having an amino acid sequence comprising the whole or a part of the amino acid sequence specified in sequence ID NO 1.
• the present invention provides a DNA encoding said RPDL protein or a variant of said RPDL protein; a vector which contains a DNA encoding said RPDL protein or a variant of said RPDL protein; a transformant having, introduced thereinto, said vector; a process for producing said RPDL protein or a variant of said RPDL protein, which comprises culturing said transformant and recovering an expression product thereof; and a polyclonal antibody or a monoclonal antibody capable of combining with said RPDL protein or a variant of said RPDL protein.
• the present invention provides a DNA probe having a DNA sequence, said DNA sequence comprising the whole or a part of the DNA sequence specified in sequence ID NO 2 or comprising a sequence complementary to the whole or a part of the DNA sequence specified in sequence ID NO 2; a DNA primer having a DNA sequence, said DNA sequence comprising a part of the DNA sequence specified in sequence ID NO 2 or comprising a sequence complementary to a part of the DNA sequence specified in sequence ID NO 2; and a gene analysis method for an RPDL protein characterized by hybridizing said DNA probe or said DNA primer with a subject DNA.
• the present invntion relates to (1) a protein comprising the whole or a part of the protein represented by sequence ID NO 1 or a variant thereof; (2) a DNA comprising the whole or a part of the DNA represented by sequence ID NO 2 or a mutant thereof, (3) a plasmid including the above DNA and a transformant carrying the same, (4) a process for producing the above protein, (5) an antibody capable of binding with the above protein, and (6) a probe or primer including a part of the above DNA sequence and a gene analysis method or gene amplification method characterized by using the same.
• cDNA was synthesized on the basis of mRNA derived from human fetal lung and a cDNA library containing cloned cDNA inserts in a given direction was prepared.
• the nucleotide sequence of each clone of this library was determined partially from the 5'-terminal side and one clone having a nucleotide sequence homologous with the RPD3 gene of a yeast was obtained. Further, the whole nucleotide sequence of this clone was determined with the result that the desired full-length cDNA sequence was obtained.
• sequence ID NO 1 The present inventors designated the protein having the sequence specified in sequence ID NO 1 as a RPDL protein, this designation being employed throughout this description.
• the DNA of the present invention and a DNA complementary to said DNA can find applications in gene and gene expression analyses by the use of a part thereof as a primer or probe.
• a part of the DNA as used herein means a sequence of continuous at least six nucleotides, preferably at least eight nucleotides, still more preferably at least ten nucleotides, and most preferably 10 to 12 nucleotides or 15 to 25 nucleotides corresponding to (i.e., contained in or complementary to) the nucleotide sequence of the DNA according to the present invention.
• the primer or probe of the present invention which is an oligonucleotide or polynucleotide may contain also at least one nucleotide(s) not corresponding to the nucleotide sequence of the DNA encoding the RPDL protein.
• the protein of the present invention can find applications in antibody preparation and agents for study and diagnosis containing such antibodies by the use of the whole or a part thereof as an epitope.
• epitope means an antigenic determinant of a polypeptide. It is well known that the epitope is generally composed of at least 5 amino acid residues and that a polypeptide composed of 6 amino acid residues combines with an antibody [see WO of PCT Patent Applications No. 8403564, published on Sep. 13, 1984 (Assignee: COMMONWEALTH SERUM LABS AND GEYSEN, H.M.)].
• a part of the protein refers to a polypeptide comprising at least about 3 to 5 consecutive amino acid residues, preferably at least about 8 to 10 consecutive amino acid residues, and still more preferably at least about 11 to 20 consecutive amino acid residues on the basis of the amino acid sequence of the protein of the present invention. Needless to say, use can be made of even a polypeptide compring at least about 20 amino acid residues.
• the polypeptide described above may contain also at least one amino acid residues not corresponding to the amino acid sequence of the RPDL protein.
• the present invention comprehends RPDL proteins which are substantially equivalent to the RPDL protein having an amino acid sequence specified in sequence ID NO 1 and which are obtained by addition, deletion, insertion or substitution of one or more constituent amino acid residues of the above protein.
• Such equivalents are included in the present invention as long as they exert similar effects in the study and diagnosis regarding the RPDL protein.
• DNAs which are substantially equivalent to the DNA encoding the RPDL protein having an amino acid sequence specified in sequence ID NO 1 and which are obtained by addition, deletion, insertion or substitution of one or more constituent nucleotides of the above DNA, i.e., equivalents, are also included in the present invention.
• a transformant can be obtained by inserting the DNA of the present invention or a part thereof into a suitable vector and transfecting this vector into suitable host cells.
• Human RPDL protein or a part thereof can be produced in a large quantity by culturing the transformant in the customary manner and separating from the resultant culture.
• a recombinant expression vector can be prepared by religating the above DNA or a fragment thereof to a vector suitable for the expression downstream of the promoter according to the customary procedure in which a restriction enzyme and DNA ligase are employed.
• Suitable vectors include plasmids pBR322 and pUC18 derived from Escherichia coli, plasmid pUB110 derived from Bacillus subtilis, plasmid pRB15 derived from yeast, bacteriophage vectors ⁇ gt10 and ⁇ gt11, and vector SV40.
• the vectors are not particularly limited as long as they can be replicated or amplified in the host.
• the promoter and terminator are also not particularly limited as long as they suit the host employed in the expression of the DNA sequence. Appropriate members thereof can be used in combination in accordance with the host.
• the DNA to be employed is not limited to the one having a DNA sequence specified in sequence ID NO 2.
• Use may be made of a DNA having a DNA sequence resulting from intentional or unintentional substitution, deletion, insertion and/or addition conducted individually or in combination at a part of the DNA sequence of sequence ID NO 2. Further, use may be made of one chemically synthesized.
• the obtained recombinant expression vector is introduced into a host in accordance with any of the competent cell method [see J. Mol. Biol., Vol.53, p.154 (1970)], the protoplast method [see Proc. Natl. Acad. Sci. USA, Vol.75, p.1929 (1978)], the calcium phosphate method [see Science, Vol.221, p.551 (1983)], the in vitro packaging method [see Proc. Natl. Acad. Sci. USA, Vol.72, p.581 (1975)], the virus vector method [see Cell, Vol.37, p.1053 (1984)], etc., thereby preparing a transformant.
• the competent cell method see J. Mol. Biol., Vol.53, p.154 (1970)
• the protoplast method see Proc. Natl. Acad. Sci. USA, Vol.75, p.1929 (1978)
• the calcium phosphate method see Science, Vol.221, p.551 (1983)
• the obtained transformant is cultured in a medium suitable for the host.
• the culturing is generally conducted at 20 to 45°C and at pH of 5 to 8, in which aeration and agitation are executed according to necessity.
• the separation of the RPDL protein from the resultant culture and its purification may be conducted by an appropriate combination of conventional separation and purification methods. Examples of these conventional methods include salting out, solvent precipitation, dialysis, gel filtration, electrophoresis, ion exchange chromatography, affinity chromatography, and reversed-phase high-performance liquid chromatography.
• Antibodies can be prepared by the conventional method in which the whole or a part of the RPDL protein is used as an antigen.
• a polyclonal antibody can be prepared by giving a plurality of subcutaneous, intramuscular, intraperitoneal or intravenous inoculations of the antigen to an animal such as a mouse, a guinea-pig and a rabbit to thereby satisfactorily immunize the same, collecting the blood specimen from the animal, and performing serum separation.
• commercially available adjuvants can be used.
• a monoclonal antibody can be prepared by, for example, conducting the fusion of splenocytes of a mouse immunized with the RPDL protein with commercially available mouse myeloma cells to thereby prepare a hybridoma and either culturing the hybridoma followed by separation of the antibody from the resultant supernatant or administering the hybridoma to a mouse followed by separation of the antibody from the mouse ascites.
• the RPDL protein as an antigen does not necessarily have to possess the whole amino acid structure but use may be made of a peptide having a partial structure of the protein, a variant or derivative of the protein, or a fusion peptide resulting from the fusion with another peptide.
• the method for preparing these is not critical and it may be biological or chemosynthetic.
• the obtained antibody enables the identification and quantity determination of RPDL protein in human biospecimens and can be used in, for example, various agents.
• the immunoassay of RPDL protein may be conducted in accordance with the generally known procedure and can be executed by, for example, any of the fluorescent antibody technique, passive agglutination and enzyme antibody technique.
• Any mutation of a gene encoding the RPDL protein can be analyzed by the use of a probe comprising a restriction enzyme fragment of the DNA provided by the present invention or by the use of, as a primer, an oligonucleotide obtained by appropriately selecting a suitably positioned nucleotide sequence from the DNA and synthesizing therewith.
• any abnormality such as insertion and deletion in the gene of a specimen can be detected by the above analysis.
• the Escherichia coli L1-3977 carrying the plasmid containing the DNA encoding the above RPDL protein was deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry under the accession number FERM BP-4805 on September 21, 1994.
• the use of a substance including the whole or a part of each of the human RPDL protein and the DNA encoding the protein according to the present invention has enabled analyses on the level of human cells not only of the functions of the above protein as a transcriptional control factor and the gene per se but also of the effects of any variation of the above protein.
• the protein of the present invention is an important transcriptional control protein commonly acting on many target genes and having both functions of activation and inactivation from the viewpoint of the homology of its amino acid sequence with that of the yeast RPD3.
• the contribution of the above investigations to the elucidation of the fundamental working of human cells, such as differentiation, amplification, activity and life and death thereof, is being anticipated.
• the sequence structure of the above gene and its location on the chromosome have been defined, so that it can be anticipated that its relationships with not only tumors but also other various diseases and abnormalities of the gene are elucidated and its application is found in the pharmaceutical field.
• cDNA was synthesized on the basis of mRNA derived from the human fetal lung (purchased from Clontech) and a cDNA library containing cloned cDNA inserts in a given direction was prepared by the use of UniZAPxR vector kit (purchased from Stratagene).
• the nucleotide sequence of each of 2058 clones of the cDNA library prepared in Example 1 was partially determined from the 5'-terminal side.
• the resultant nucleotide sequences were compared with the known nucleotide sequences of a data base to find out one clone L1-3977 having homology with the yeast RPD3.
• a partial sequence (256 bp) of the clone L1-3977 exhibited a homology of 60.2% with the RPD3 gene (Accession No. S66438, 1645 bp) of yeast (Saccharomyces cerevisiae) in the range of 176 bp.
• the DNA sequence of the clone Ll-3977 obtained in Example 2 was determined by the Dideoxy method [see F. Sanger et al., Proc. Natl. Acad. Sci. USA, Vol.74, p.p.5463-5467 (1977)]. As a result, it was found that the clone L1-3977 contained a full-length cDNA having a novel sequence specified in sequence ID NO 2.
• sequence ID NO 1 sequence ID NO 1, RPDL protein
• This amino acid sequence exhibited a homology of 60.0% with the RPD3 protein (Accession No. S22284 & P32561, 433 amino acid residues) of yeast (S. cerevisiae) in the range of 422 amino acid residues.
• the nucleotide sequence of sequence ID NO 2 (2111 bp) exhibited a homology of 62.1% with the RPD3 gene (Accession No. S66438, 1645 bp) of yeast (S. cerevisiae) in the range of 1168 bp. Further, it exhibited a homology as high as 80.9% with the RPD3 homologue gene (Accession No. X78454, 1040 bp) of Xenopus laevis in the range of 1034 bp. A homology as high as 94.8% was recognized in the range of 343 amino acid residues between the protein (343 amino acid residues) encoded by the RPD3 homologue gene (Accession No. X78454, 1040 bp) of Xenopus laevis and the RPDL protein of the present invention.
• the RPDL protein of the present invention is an important human transcriptional control protein having the same functions as those of the RPD3 protein of a yeast.
• the nucleotide sequence (2111 bp) of sequence ID NO 2 has exhibited a homology as high as 78.9% with the nucleotide sequence of the 3'-noncoding region of proto-oncogene c-tk1 (chicken tyrosine kinase proto-oncogene) in a range as wide as 1534 bp, so that the importance of the RPDL protein of the present invention in the transcriptional control mechanism has also been supported from the recent information on the association of the gene 3'-noncoding region with the control of transcription and translation.
• the cDNA obtained in Example 3 was used as a probe for investigating the location of the gene encoding the RPDL protein of the present invention on the chromosome. That is, the location on the chromosome with which the above probe hybridized was determined by the FISH method [see Inazawa et al., Genomics, Vol.10, p.p.1075-1078 (1991)]. As a result, the location was identified as being at 1p34.1 on the short arm of the chromosome 1. This location was the one at which deletion was recognized in mammary carcinoma [see A. Borg et al., Genes Chromosome Cancer, Vol.5, p.p.311-320 (1992)] and gastric carcinoma [see T. Sano et al., Cancer Res., Vol.51, p.p.2926-2931 (1991)].
• a partial sequence including the protein coding region was amplified by the PCR with the use of the cDNA obtained in Example 3 as a template. BamHI and EcoRI cleavage sites were added to the 5'-terminus of one primer and the 5'-terminus of the other primer, respectively. The obtained amplification product was digested with BamHI and EcoRI. The resultant fragment was inserted into expression vector pGEX-2T (purchased from Pharmacia) preliminaly digested with BamHI and EcoRI, thereby constructing expression plasmid pGST-RPDL.
• pGEX-2T purchasedd from Pharmacia
• coli DH5 ⁇ was transformed with the plasmid pGST-RPDL and resulting transformants were selected based on the ampicillin resistance, thereby obtaining a transformant capable of expressing a fusion protein of glutathione-S-transferase and RPDL protein.
• Example 6 The transformant obtained in Example 6 was cultured, and a recombinant RPDL fusion protein was extracted from the resultant culture and purified.
• the transformant was cultured by shaking the same in 100 ml of LB medium (1% peptone, 0.5% yeast extract and 1% NaCl) at 37°C overnight.
• the resultant liquid culture was diluted tenfold with LB medium preliminaly heated to 37°C and the resulting dilution was further cultured at 37°C for 30 to 90 minutes, thereby obtaining a culture of logarithmic growth phase.
• Isopropyl ⁇ -D-thiogalactopyranoside was added to 1 l of the culture so that the final concentration thereof was 1 mM, followed by culturing for 3 to 4 hours. The culture was centrifuged to thereby separate bacterial cells.

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• 1995
  • 1995-09-14 US US08/528,255 patent/US5659016A/en not_active Expired - Lifetime
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• 1996
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